It has been established that mammalian cytochrome a3 has two low potential forms in addition to a high potential form described in last year's report. Both low potential forms have an alpha peak at 602 nm, but are distinguished by their Soret peak; one at 429 nm and one at 448 nm. The a3(429) is thermodynamically more stable than the a3(448) nm but it is formed very slowly unless three requirements are met. There must be extra lipids and proteins present (i.e. egg homogenate), K3Fe(CN)6 at -0.5 mM, and an exposure to -400 mV for 1 hr. If the enzyme is incorporated into a proteoliposome membrane, the requirement for egg homogenate is removed. Therefore, the a3(429) form must represent the biologically significant species. Both forms are capable of forming CO complexes (CO is analogous to 02). The Em's of both a3(448) and a3(429) are near 180 mV as established by both oxidative and reductive titrations. The Em of a3(448).CO is near 350 mV and the Em of a3(429).CO is near 250 mV. Both species can be oxidized using K3Fe(CN)6 as the oxidizing agent. This finding was not anticipated on the basis of past work. However, in these earlier studies, K3Fe(CN)6 was added at a very low temperature because of the concern that 02 might displace CO from the enzyme at room temperature. Our experiments were performed anaerobically at room temperature.